Minimum Inhibitory Concentration (MIC) Assay Protocol

The Biofilm-Based Minimum Inhibitory Concentration (MIC) assay is an essential method for evaluating the effectiveness of antimicrobial agents against biofilm-associated microorganisms, which exhibit heightened resistance due to their protective extracellular matrix. A crucial step in this assay is the disruption of biofilm structures to release embedded cells for accurate viability assessment. The UIP400MTP multi-well plate sonicator facilitates this process by employing focused ultrasound to generate controlled cavitation, efficiently detaching biofilm cells and dispersing them into a uniform suspension. This precise and reproducible biofilm disruption enhances the reliability and throughput of MIC assays, making the UIP400MTP an essential tool in advancing biofilm research.

Sonication for Biofilm Detachment

The Biofilm-Based MIC Assay typically measures bacterial viability or growth inhibition using methods such as plating, colony counting, or optical density measurements. Sonication is a critical step in biofilm-based MIC assays when assessing the antimicrobial susceptibility of biofilm-associated microorganisms. Its primary function is to detach and disperse cells embedded in the biofilm matrix into a uniform suspension for accurate analysis.
Biofilms are significantly more resistant to antimicrobial agents compared to planktonic cells, making proper detachment critical for accurate analysis. During this process, ultrasonic waves generate controlled cavitation, breaking apart the biofilm matrix and releasing embedded cells into a uniform suspension within the recovery medium. This step enables precise viability assessments of biofilm-dispersed cells through methods such as plating, dilution, and colony counting. Proper biofilm disruption via sonication prevents residual matrix components from shielding cells, which could otherwise lead to an underestimation of antimicrobial activity. The multi-well plate sonicator UIP400MTP is particularly well-suited for this purpose, offering precise and reproducible sonication conditions to ensure reliable and high-throughput preparation of assay plates.

Why Sonication is Necessary in Biofilm-Based Minimum Inhibitrory Concentration Assays

For viability measurements and cell counting, a complete and reliable detachment and dispersion of single cells is required. The UIP400MTP promotes a uniform, non-damaging biofilm detachment and cell dispersion for robust assay results.

• Biofilm Complexity: Biofilms are structured microbial communities encased in an extracellular polymeric substance (EPS) matrix, which protects the microorganisms and makes them more resistant to antimicrobial agents.
• Uniform Dispersion: To accurately measure the viability of biofilm-embedded cells or their susceptibility to antimicrobials, the biofilm must first be dislodged and broken down into a homogeneous suspension.

Biofilm-Based Minimum Inhibitory Concentration Assay Protocol

The Minimum Inhibitory Concentration (MIC) assay determines the lowest concentration of an antimicrobial agent required to inhibit the visible growth of microorganisms. This protocol is designed for biofilm-associated microorganisms, using UIP400MTP multi-well plate sonicator for biofilm disruption.

Step 1: Preparation of Bacterial Inoculum

1. Prepare the bacterial suspension:
   Grow bacteria in appropriate media to the mid-logarithmic phase.
   Dilute the culture to achieve a standardized cell density (e.g., 0.5 McFarland standard or OD600 ~0.1).
2. Prepare antimicrobial solutions:
   Dilute the antimicrobial agent in a suitable medium to create a range of concentrations (e.g., twofold serial dilutions).
3. Dispense into the 96well plate:
   Add the antimicrobial solutions into the wells of a standard 96-well plate, with a final well volume of ~150–200 µL.
   Include growth controls (no antimicrobial) and sterility controls (no bacterial inoculum).

Step 2: Biofilm Formation on Peg Lid

• Attach the peg lid:
  Place the specialized peg lid onto the inoculated wells, ensuring the pegs are fully submerged in the bacterial suspension.
• Incubate the plate:
  Incubate at the appropriate temperature (e.g., 37°C) for a specified duration (e.g., 24 hours) under static conditions to allow biofilm formation on the pegs.

Step 3: Antimicrobial Exposure

• Rinse the pegs:
  Remove the peg lid from the bacterial suspension and gently rinse in sterile saline or PBS to remove loosely attached planktonic cells.
• Expose to antimicrobials:
  Transfer the peg lid into a new 96well plate containing the antimicrobial dilutions prepared earlier.
  Incubate for a defined period (e.g., 24 hours) under static conditions to allow the antimicrobial agent to act on the biofilms.

Step 4: Sonication with the Microplate Sonicator UIP400MTP

The sonication step is critical for detaching biofilms from the peg lids to assess viability. Follow these steps for the UIP400MTP sonicator:

1. Prepare the setup:
   Fill a fresh 96-well plate with recovery medium (e.g., neutralizing broth or sterile growth medium) in each well.
2. Transfer the peg lid:
   Remove the peg lid from the antimicrobial treatment plate.
   Rinse the peg lid in sterile saline or PBS to remove residual antimicrobial agents.
3. Position the plate in the sonicator:
   Attach the peg lid to the recovery medium plate.
   Place the recovery medium plate into the UIP400MTP sonicator, ensuring the plate sits centered and stable as in the manual described.
4. Adjust sonication parameters:
   Set the sonication parameters at the UIP400MTP (settings may be adjusted to the biofilm):
   Amplitude: 70–100%.
   Sonication time: 1–3 minutes (adjust based on biofilm structure) at cycle mode.
5. Sonicate:
   Start the sonication process. The ultrasonic waves will disrupt the biofilm matrix and dislodge the cells into the recovery medium.
6. Monitor the process:
   Use the pluggable temperature sensor to monitor the sample temperature in the wells. The UIP400MTP can be connected to a lab chiller for cooling.
7. Post-sonication handling:
   Immediately transfer the recovery medium containing detached biofilms into a fresh sterile plate for subsequent analysis.

(A) Plate containing TSB with 2% glucose used for biofilm formation, cell recovery, and determination of MIC and MBEC; (B) Lid with pins for formation of staphylococcal biofilms.
The biofilm cells formed on the pins were dislodged by sonication (Hielscher Ultrasound Technology) for 5 min in 96-well plates containing fresh culture medium for recovery of the cells.
(Picture and study: ©de Oliveira et al., 2016)

Step 4: Viability Assessment

Plate and culture detached biofilms:

1. Perform serial dilutions of the recovery medium and plate onto agar to enumerate colony-forming units (CFU).
2. Evaluate MIC:
   Determine the MIC as the lowest antimicrobial concentration that completely inhibits visible microbial growth in the recovery medium.

MIC Minimum Inhibitory Concentration: Example of microtiter plate and interpretation of microdilution results.
(Study and image: ©Jaskiewicz et al. 2019)

Design, Manufacturing and Consulting – Quality Made in Germany

Hielscher ultrasonicators are well-known for their highest quality and design standards. Robustness and easy operation allow the smooth integration of our ultrasonicators into industrial facilities. Rough conditions and demanding environments are easily handled by Hielscher sonicators.

Hielscher Ultrasonics is an ISO certified company and put special emphasis on high-performance ultrasonicators featuring state-of-the-art technology and user-friendliness. Of course, Hielscher ultrasonicators are CE compliant and meet the requirements of UL, CSA and RoHs.

Read here how the UIP400MTP is used for biofilm detachment in the ASTM E2799 protocol – Testing disinfectant efficacy against Pseudomonas aeruginosa biofilm using the MBEC assay.